Intracellular Soluble Protein Research Articles

Active substances of myxobacteria against plant diseases and their action mechanisms.

Myxobacteria have a complex life cycle and unique social behavior, and obtain nutrients by preying on bacteria and fungi in soil. Chitinase, β-1,3 glucanase and β-1,6 glucanase produced by myxobacteria can degrade the glycosidic bond of cell wall of some plant pathogenic fungi, resulting in a perforated structure in the cell wall. In addition, isooctanol produced by myxobacteria can lead to the accumulation of intracellular reactive oxygen species in some pathogenic fungi and induce cell apoptosis. Myxobacteria can also perforate the cell wall of some plant pathogenic oomycetes by β-1,3 glucanase, reduce the content of intracellular soluble protein and protective enzyme activity, affect the permeability of oomycete cell membrane, and aggravate the oxidative damage of pathogen cells. Small molecule compounds such as diisobutyl phthalate and myxovirescin produced by myxobacteria can inhibit the formation of biofilm and lipoprotein of bacteria, and cystobactamids can inhibit the activity of DNA gyrase, thus changing the permeability of bacterial cell membrane. Myxobacteria, as a new natural compound resource bank, can control plant pathogenic fungi, oomycetes and bacteria by producing carbohydrate active enzymes and small molecular compounds, so it has great potential in plant disease control.

Nanomaterized zinc sulfide-meerschaum biomatrix efficiently suppressed Fusarium verticilloides with augmented rice seed quality benefits during storage.

The seed-borne mycopathogen Fusarium verticilliodes is a serious and deleterious pathogen causing substantial losses of rice seeds and grains. Rice seeds are prone to infestation at all points of the production chain and the fungal pathogen continues dormant devastation even during storage, adversely affecting the seed parameters. Its control is compromised due to the nonavailability of recommended fungicides during storage. Nanotechnological interventions can provide effective and ecofriendly alternative against mycopathogens during resting periods. Herein, the zinc sulfide-meerschaum nano bio-matrix (nZnS-MR) is presented to show this strategy, which worked well against F. verticilliodes when applied on freshly harvested rice seeds during 6 months of storage. The healthy, smooth and rounded girths of F. verticilloides mycelium were reduced with loss of turgidity, disrupting the hyphal exterior architecture, during in vitro treatment with nZnS-MR, endorsed by staining methodology, crystal violet and intracellular soluble protein leakage assays. In vivo application on rice seeds optimized 750 μg g-1 of nano zinc sulfide (nZnS) for 6 months of application during storage with maximum reduction of disease parameters [seedling blight (1.19%) and seed rot (5.43%)] and most augmented quality parameters [maximum germination (94.14%), seedling length (22.50 cm), dry weight (0.121 g) and vigor index (11.37)]. nZnS-MR acted as a slow release nanoformulation of nZnS for long-term antifungal activity. nZnS-MR is presented as an ecofriendly, biocompatible, bio-efficient, profertilization, cost-effective green material for the control of F. verticilliodes with rice seed invigorating effect, describing it as new a nano-generation material for efficient storage application.

Glucose-Limited Fed-Batch Cultivation Strategy to Mimic Large-Scale Effects in Escherichia coli Linked to Accumulation of Non-Canonical Branched-Chain Amino Acids by Combination of Pyruvate Pulses and Dissolved Oxygen Limitation

Insufficient mixing in large-scale bioreactors provokes gradient zones of substrate, dissolved oxygen (DO), pH, and other parameters. E. coli responds to a high glucose, low oxygen feeding zone with the accumulation of mixed acid fermentation products, especially formate, but also with the synthesis of non-canonical amino acids, such as norvaline, norleucine and β-methylnorleucine. These amino acids can be mis-incorporated into recombinant products, which causes a problem for pharmaceutical production whose solution is not trivial. While these effects can also be observed in scale down bioreactor systems, these are challenging to operate. Especially the high-throughput screening of clone libraries is not easy, as fed-batch cultivations would need to be controlled via repeated glucose pulses with simultaneous oxygen limitation, as has been demonstrated in well controlled robotic systems. Here we show that not only glucose pulses in combination with oxygen limitation can provoke the synthesis of these non-canonical branched-chain amino acids (ncBCAA), but also that pyruvate pulses produce the same effect. Therefore, we combined the enzyme-based glucose delivery method Enbase® in a PALL24 mini-bioreactor system and combined repeated pyruvate pulses with simultaneous reduction of the aeration rate. These cultivation conditions produced an increase in the non-canonical branched chain amino acids norvaline and norleucine in both the intracellular soluble protein and inclusion body fractions with mini-proinsulin as an example product, and this effect was verified in a 15 L stirred tank bioreactor (STR). To our opinion this cultivation strategy is easy to apply for the screening of strain libraries under standard laboratory conditions if no complex robotic and well controlled parallel cultivation devices are available.

Co-expressing GroEL\u2013GroES, Ssa1\u2013Sis1 and Bip\u2013PDI chaperones for enhanced intracellular production and partial-wall breaking improved stability of porcine growth hormone

Porcine growth hormone (pGH) is a class of peptide hormones secreted from the pituitary gland, which can significantly improve growth and feed utilization of pigs. However, it is unstable and volatile in vitro. It needs to be encapsulated in liposomes when feeding livestock, whose high cost greatly limits its application in pig industry. Therefore we attempted to express pGH as intracellular soluble protein in Pichia pastoris and feed these yeasts with partial wall-breaking for swine, which could release directly pGH in intestine tract in case of being degraded in intestinal tract with low cost. In order to improve the intracellular soluble expression of pGH protein in Pichia pastoris and stability in vitro, we optimized the pGH gene, and screened molecular chaperones from E. coli and Pichia pastoris respectively for co-expressing with pGH. In addition, we had also explored conditions of mechanical crushing and fermentation. The results showed that the expression of intracellular soluble pGH protein was significantly increased after gene optimized and co-expressed with Ssa1–Sis1 chaperone from Pichia pastoris. Meanwhile, the optimal conditions of partial wall-breaking and fermentation of Pichia pastoris were confirmed, the data showed that the intracellular expression of the optimized pGH protein co-expressed with Ssa1–Sis1 could reach 340 mg/L with optimal conditions of partial wall-breaking and fermentation. Animal experiments verified that the optimized pGH protein co-expression with Ssa1–Sis1 had the best promoting effects on the growth of piglets. Our study demonstrated that Ssa1–Sis1 could enhance the intracellular soluble expression of pGH protein in Pichia pastoris and that partial wall-breaking of yeast could prevent pGH from degradation in vitro, release targetedly in the intestine and play its biological function effectively. Our study could provide a new idea to cut the cost effectively, establishing a theoretical basis for the clinic application of unstable substances in vitro.

( E)-2-Hexenal, as a Potential Natural Antifungal Compound, Inhibits Aspergillus flavus Spore Germination by Disrupting Mitochondrial Energy Metabolism.

Fungal contamination imposes threats to agriculture and food production and human health. A method to safely and effectively restrict fungal contamination is still needed. Here, we report the effect and mode of action of ( E)-2-hexenal, one of the green leaf volatiles (GLVs), on the spore germination of Aspergillus flavus, which can contaminate a variety of crops. The EC50 value, minimum inhibitory concentration (MIC), and minimum fungicidal concentration (MFC) of ( E)-2-hexenal were 0.26, 1.0, and 4.0 μL/mL, respectively. As observed by scanning electron microscopy (SEM), the surface morphology of A. flavus spores did not change after treatment with the MIC of ( E)-2-hexenal, but the spores were shrunken and depressed upon treatment with the MFC of ( E)-2-hexenal. The MIC and MFC of ( E)-2-hexenal induced evident phosphatidylserine (PS) externalization of A. flavus spores as detected by double staining with Annexin V-FITC and propidium iodide, indicating that early apoptosis was potentially induced. Furthermore, sublethal doses of ( E)-2-hexenal disturbed pyruvate metabolism and reduced the intracellular soluble protein content of A. flavus spores during the early stage of germination, and MIC treatment decreased acetyl-CoA and ATP contents by 65.7 ± 3.7% and 53.9 ± 4.0% ( P < 0.05), respectively. Additionally, the activity of mitochondrial dehydrogenases was dramatically inhibited by 23.8 ± 2.2% ( P < 0.05) at the MIC of ( E)-2-hexenal. Therefore, the disruption of mitochondrial energy metabolism and the induction of early apoptosis are involved in the mechanism of action of ( E)-2-hexenal against A. flavus spore germination.

Scaling-up fermentation of Escherichia coli for production of recombinant P64k protein from Neisseria meningitidis

Background: P64k is a Neisseria meningitidis high molecular weight protein present in meningococcal vaccine preparations. The lpdA gene, which encodes for this protein, was cloned in Escherichia coli and the P64k recombinant protein was expressed in E. coli K12 GC366 cells under the control of a tryptophan promoter. P64k was expressed as an intracellular soluble protein about 28% of the total cellular protein. Several scale-up criteria of fermentation processes were studied to obtain the recombinant P64k protein at the pilot production scale. Results: The best operational conditions at a larger scale production of P64k recombinant protein were studied and compared using the four following criteria: Constant Reynold's number (Re constant), Constant impeller tip speed (n di constant), Constant power consumption per unit liquid volume (P/V constant) and Constant volumetric oxygen transfer coefficients (KLa/k constant). The highest production of the recombinant protein was achieved based on the constant KLa/k scale-up fermentation criterion, calculating the aeration rate (Q) and the impeller agitation speed (n) by iterative process, keeping constant the KLa/k value from bench scale. The P64k protein total production at the 50 l culture scale was 546 mg l-1 in comparison with the 284 mg l-1 obtained at 1.5 l bench scale. Conclusions: The methodology described herein, for the KLa/k scale-up fermentation criterion, allowed us to obtain the P64k protein at 50 l scale. A fermentation process for the production of P64k protein from N. meningitidis was established, a protein to be used in future vaccine formulations in humans.

Scaling-up fermentation of Escherichia coli for production of recombinant P64k protein from Neisseria meningitidis

P64k is a Neisseria meningitidis high molecular weight protein present in meningococcal vaccine preparations. The lpdA gene, which encodes for this protein, was cloned in Escherichia coli and the P64k recombinant protein was expressed in E. coli K12 GC366 cells under the control of a tryptophan promoter. P64k was expressed as an intracellular soluble protein about 28% of the total cellular protein. Several scale-up criteria of fermentation processes were studied to obtain the recombinant P64k protein at the pilot production scale. The best operational conditions at a larger scale production of P64k recombinant protein were studied and compared using the four following criteria: Constant Reynold's number (Re constant), Constant impeller tip speed (n di constant), Constant power consumption per unit liquid volume (P/V constant) and Constant volumetric oxygen transfer coefficients (KLa/k constant). The highest production of the recombinant protein was achieved based on the constant KLa/k scale-up fermentation criterion, calculating the aeration rate (Q) and the impeller agitation speed (n) by iterative process, keeping constant the KLa/k value from bench scale. The P64k protein total production at the 50 l culture scale was 546 mg l-1 in comparison with the 284 mg l-1 obtained at 1.5 l bench scale. The methodology described herein, for the KLa/k scale-up fermentation criterion, allowed us to obtain the P64k protein at 50 l scale. A fermentation process for the production of P64k protein from N. meningitidis was established, a protein to be used in future vaccine formulations in humans. How to cite: Espinosa R, García J, Narciandi E, et al. Scaling-up fermentation of Escherichia coli for production of recombinant P64k protein from Neisseria meningitidis. Electron J Biotechnol 2018;33. https://doi.org/10.1016/j.ejbt.2018.03.004.

Purification, Characterization, and Cloning of a Cold-Adapted Protease from Antarctic Janthinobacterium lividum.

In this study, a 107 kDa protease from psychrophilic Janthinobacterium lividum PAMC 26541 was purified by anion-exchange chromatography. The specific activity of the purified protease was 264 U/mg, and the overall yield was 12.5%. The J. lividum PAMC 25641 protease showed optimal activity at pH 7.0-7.5 and 40°C. Protease activity was inhibited by PMSF, but not by DTT. On the basis of the N-terminal sequence of the purified protease, the gene encoding the cold-adapted protease from J. lividum PAMC 25641 was cloned into the pET-28a(+) vector and heterologously expressed in Escherichia coli BL21(DE3) as an intracellular soluble protein.

Biochemical Characterization of a Novel Acidic Exochitinase from Rhizomucor miehei with Antifungal Activity.

A novel chitinase gene (RmChi44) from Rhizomucor miehei was cloned and expressed in Escherichia coli as an intracellular soluble and active protein. The recombinant chitinase (RmChi44) was purified to homogeneity and biochemically characterized. The molecular mass of RmChi44 was estimated to be 44.6 kDa on SDS-PAGE. RmChi44 displayed an acidic pH optimum of 4.5 and was stable within pH 4.5-9.0. The optimal temperature of RmChi44 was found to be 50 °C. The Km values of RmChi44 for colloidal chitin and glycol chitin were 4.02 and 1.55 mg/mL, respectively. RmChi44 hydrolyzed colloidal chitin to yield mainly N-acetyl chitobiose, exhibiting an exotype cleavage pattern. Moreover, the enzyme displayed β-N-acetylglucosaminidase activity, splitting N-acetyl COSs with degree of polymerization (DP) 2-5 into their monomer. In addition, RmChi44 showed antifungal activity against some phytopathogenic fungi. This is the first report on an exochitinase showing β-N-acetylglucosaminidase activity and antifungal activity from Rhizomucor species.

Gene cloning, functional expression and characterisation of a novel type I pullulanase from Paenibacillus barengoltzii and its application in resistant starch production

A novel pullulanase gene (PbPulA) from Paenibacillus barengoltzii was cloned. PbPulA has an open reading frame of 2028 bp encoding 675 amino acids. It was heterologously expressed in Escherichia coli as an intracellular soluble protein. The recombinant pullulanase (PbPulA) was purified to homogeneity with a molecular mass of about 75 kDa on SDS-PAGE. PbPulA was optimally active at pH 5.5 and 50 °C. It was stable within pH 5.5–10.5. The enzyme exhibited strict substrate specificity towards pullulan, but showed relatively low activity towards amylopectin and no activity towards other tested polysaccharides. The Km and Vmax values of the enzyme on pullulan were 2.94 mg/mL and 280.5 μmol/min/mg, respectively. The addition of PbPulA in gelatinized rice and maize starches significantly increased the resistant starch type 3 (RS3) yields. Final yields from rice and maize starches were 10.82 g/100 g and 11.41 g/100 g, respectively. These properties make PbPulA useful in starch industries.

An acidic, thermostable exochitinase with β-N-acetylglucosaminidase activity from Paenibacillus barengoltzii converting chitin to N-acetyl glucosamine.

BackgroundN-acetyl-β-D-glucosamine (GlcNAc) is widely used as a valuable pharmacological agent and a functional food additive. The traditional chemical process for GlcNAc production has some problems such as high production cost, low yield, and acidic pollution. Hence, to identify a novel chitinase that is suitable for bioconversion of chitin to GlcNAc is of great value.ResultsA novel chitinase gene (PbChi74) from Paenibacillus barengoltzii was cloned and heterologously expressed in Escherichia coli as an intracellular soluble protein. The gene has an open reading frame (ORF) of 2,163 bp encoding 720 amino acids. The recombinant chitinase (PbChi74) was purified to apparent homogeneity with a purification fold of 2.2 and a recovery yield of 57.9%. The molecular mass of the purified enzyme was estimated to be 74.6 kDa and 74.3 kDa by SDS-PAGE and gel filtration, respectively. PbChi74 displayed an acidic pH optimum of 4.5 and a temperature optimum of 65°C. The enzyme showed high activity toward colloidal chitin, glycol chitin, N-acetyl chitooligosaccharides, and p-nitrophenyl N-acetyl β-glucosaminide. PbChi74 hydrolyzed colloidal chitin to yield N-acetyl chitobiose [(GlcNAc)2] at the initial stage, which was further converted to its monomer N-acetyl glucosamine (GlcNAc), suggesting that it is an exochitinase with β-N-acetylglucosaminidase activity. The purified PbChi74 coupled with RmNAG (β-N-acetylglucosaminidase from Rhizomucor miehei) was used to convert colloidal chitin to GlcNAc, and GlcNAc was the sole end product at a concentration of 27.8 mg mL-1 with a conversion yield of 92.6%. These results suggest that PbChi74 may have great potential in chitin conversion.ConclusionsThe excellent thermostability and hydrolytic properties may give the exochitinase great potential in GlcNAc production from chitin. This is the first report on an exochitinase with N-acetyl-β-D-glucosaminidase activity from Paenibacillus species.

Identification and analysis of the salt tolerant property of AHL lactonase (AiiATSAWB) of Bacillus species

Bacterial biofilms communicate by a process called Quorum Sensing. Gram negative bacterial pathogens specifically talk through the production, detection, and response to the signal or autoinducer called Acyl Homoserine Lactones. Bacterial lactonases are important AHL hydrolysing or quorum quenching enzymes. The present study deals with ten endospore forming gram positive isolates of the saltern soil. Preliminary screening for Quorum Quenching activity with the QS Inhibition indicator strain Chromobacterium violaceum ATCC 12472, showed positive activity in four isolates namely TS2, TS16, TSAWB, and TS53B. AHL lactonase (AiiA) specific primers amplified Acyl Homoserine Lactone lactonase gene in the TSAWB genome alone. Phylogenetic relationship of the identified AiiATSAWB confirmed its evolutionary relationship with bacterial AiiA like AHL lactonase of the metallo-beta-lactamase super family. Our in vitro AHL hydrolysis assay under wide percentage (0-5) of salt solutions with TSAWB isolate and also its intracellular soluble protein fraction showed halotolerant AHL hydrolysis ability of the AiiATSAWB enzyme. In silico determination of putative tertiary structure, the ESBRI derived conserved salt bridges, aminoacid residue characterization with high mole percent of acidic and hydrophobic residues reaffirmed the halotolerant ability of the enzyme. So we propound the future use of purified AiiATSAWB , as hypertonic suspension for inhalation to substitute the action of inactivated host's paraoxonase in treating Pseudomonas aeruginosa infection in cystic fibrosis patients.

Potassium silicate has a better mitigating effect than Potassium Nitrate on Salt Stress of Kentucky Bluegrass (Poa pratensis)

Potassium or silicon could enhance salt tolerance of plants. However there is little information about the combined effects of these two elements on plants under stress. Effects of KNO3, K2SiO3 with K+ removal or K2SiO3 on NaCl tolerance of Poa pratensis were compared. Leaf gas exchange, osmotic adjustment substances, anti-oxidative enzyme activities and biomass were compared between different treatments. It showed that the addition of KNO3 or K2SiO3 could alleviate salt stress by promoting growth; reducing intracellular proline, soluble protein and soluble sugar; increasing chlorophyll content, photosynthetic rate, water use efficiency and antioxidative enzyme activities. K2SiO3 had a better mitigation than KNO3 when the K+ concentration was the same. At the same Si concentration, the mitigating effect of K2SiO3 was not significantly different from that of K2SiO3 with removal of K+. The results indicated that there was no synergistic effect between Si and K.

Histocytological analysis of yam (Dioscorea alata) shoot tips cryopreserved by encapsulation–dehydration.

In this work, we performed qualitative and quantitative observations of the cytological changes occurring in cells of yam (Dioscorea alata) in vitro shoot tips cryopreserved using the encapsulation-dehydration (E-D) technique. Shoot tip osmoprotection for 24 h in 1.25 M sucrose medium induced drastic changes in cellular cytological features, including high plasmolysis in all three cellular areas studied, the external cell layer (L1), one to three (L1-3) and seven to nine (L7-9) cell layers from the surface of the meristematic dome, pyknotic nuclei in meristematic area cells and disappearance of nucleoli. Nucleus size decreased significantly in all cellular areas studied. Nucleocytoplasmic ratio decreased significantly in L1-3 and L7-9 cells. Nuclear protein content increased, particularly in L1 and L1-3 cells. After physical dehydration, plasma membrane of numerous basal part cells was broken and intracellular soluble protein leakage was observed. Nucleus area and nucleocytoplasmic ratio decreased significantly in L7-9 cells. One week after cryopreservation, shoot tips showed regrowth and living cells had recovered their original morphology. In all cellular areas studied, nuclei had retrieved their original staining and nucleoli were visible. Original nucleus area values were recovered in L1-3 and L1 cells. The nucleocytoplasmic ratio retrieved its initial value in L1 cells but remained at levels observed after osmoprotection for L1-3 and L7-9 cells. The nuclear protein content had retrieved its original level. This investigation provided new insights in changes occurring in D. alata apices throughout an E-D protocol.

Purification and characterization of a novel chitinase gene from Paecilomyces thermophila expressed in Escherichia coli

A novel chitinase gene ( PtChiA) from the thermophilic fungus Paecilomyces thermophila was cloned and expressed in Escherichia coli as an intracellular soluble protein. The gene sequence alignment indicates that PtChiA belongs to glycoside hydrolase (GH) family 18 and has an open reading frame comprising of 1473 bp nucleotide sequences with five introns. PtChiA encodes 400 amino acids without any predicted signal peptide. PtChiA was purified by Ni-IDA chromatography. It displayed an acidic optimum pH of 4.5 and broad pH stability (pH 4.0–10.5). The enzyme exhibited an optimal temperature of 50 °C and was stable up to 40 °C. PtChiA was strongly inhibited by anionic detergent SDS, and also by metal ions Hg 2+ and Mn 2+. It did not exhibit any antifungal activity against pathogenic fungi. It has the ability to hydrolyze colloidal chitin into chito-oligomers suggesting its use in conversion of chitin waste into chito-oligosaccharides.

Interactions of cytostatic unsaturated ketonucleo-sides with sulfhydryl containing cell constituents

The cytostatic unsaturated ketonucleosides, 1, 2, 3 and 4 are highly reactive sulfhydryl blocking agents. Kinetics of their reactions with reduced glutathione (GSH) were measured and their reactivity was compared to that of N-ethylmaleimide (NEM), acrylonitrile and chloroacetamide. Their reaction products with N-acetyl-L-cysteine (AcCys) were prepared and characterized by chemical analysis and nuclear magnetic resonance (NMR) spectroscopy. Compounds 1, 2 and 3 gave Michael type 1:1 addition products. Compound 4 reacted with AcCys by a three step mechanism; the primary addition product 8 underwent an unusual elimination reaction giving the unsaturated compound 9, which yielded the addition product 10 with AcCys. In the reaction with GSH, compound 4 behaved like a bifunctional SH alkylating agent. Compounds 1, 2, 3 and 4 also reacted with protein thiols as shown by their ability to inhibit lactate dehydrogenase (LDH). Unsaturated ketonucleosides had diversified effect on L1210 leukemia cells. While the most potent cytostatics, compounds 1 and 3, reduced considerably the membrane surface SH level, they were without effect on soluble intracellular protein thiols. In contrast, nucleosides 2 and 4, less active than the former, only slightly affected the membrane surface sulfhydryls and considerably depleted the intracellular soluble protein thiols. Only slight differences were found between the reactions of the four nucleosides with non-protein SH (NPSH). The correlation found between in vivo biological activity and cell membrane impairment suggests that selective alkylation of certain key membrane thiols by unsaturated ketonucleosides might be an important event in their biological effect.

Uricase of Bacillus fastidiosus properties and regulation of synthesis

Uricase (urate:oxygen oxidoreductase, EC 1.7.3.3) of Bacillus fastidiosus was purified to homogeneity in a two-step procedure and was crystallized. the native molecule had a molecular weight of 145 000–150000 and was composed of subunits of two kinds ( M r = 36 000 and 39 000) in a 1: 1 ratio. The quaternary structure of the enzyme was reversibly altered, with concomitant loss of activity, at temperatures between 40 and 60°C. No evidence was found for the involvement of metal ions or coenzymes in the uricase reaction. The enzyme was inhibited by various metal ions and by cyanide. The isoelectric point of the enzyme was 4.3, the pH optimum 9.5 and the optimal temperature 30–35°C. Only uric acid was oxidized by the enzyme and 9-methyluric acid, xanthine, 8-azaxanthine and oxonic acid were competitive inhibitors. Uricase synthesis was repressed by allontoin and allantoate, even in the presence of uric acid, which induced synthesis of the enzyme. Molecular oxygen was an important environmental factor in the contorl of uricase synthesis, probably due to its effect, as cosubstrate in the uricase reaction, in assessing the cytoplasmic concentration of allantoin. The highest amounts of uricase, up to half of the intracellular soluble protein content, was found in cells growing under limited oxygen supply in media containing uric acid as the main substrate.

Ligandinuria in nephrotoxic acute tubular necrosis

Ligandin (GSH-S-transferase B), an abundant intracellular soluble protein in rat proximal tubules, hepatocytes, and small intestinal mucosal cells, is believed to be a component of an organic anion transport system. Its presence in urine was determined immunologically and catalytically in adult, female, Sprague-Dawley rats that were given mercuric chloride or potassium dichromate. These nephrotoxic agents produce severe renal failure, with epithelial necrosis in the proximal tubule. Mercuric chloride perferentially injures the terminal part of the proximal tubule, while potassium dichromate damages more proximal segments. Ligandinuria was consistently detected immunologically and catalytically from 6 to 24 hr following injection of mercuric chloride. Potassium dichromate administration did not result in immunologically detectable liganduria; however, GSH-S-transferase activity, which provides a more sensitive assay, was frequently detected. The findings are consistent with immunofluorescent localization of ligandin in the proximal tubule. Immunologic or enzymatic measurement of ligandin in urine may provide a sensitive index of acute injury to the proximal tubule.

Effects of ionizing radiation upon intracellular levels of soluble microtubule protein in cultured mammalian cells

Intracellular soluble microtubule protein levels were determined in cultured mammalian cells following X-irradiation. Microtubule protein levels rose along with corresponding RNA and total soluble protein levels as cells accumulated in G 2 and declined as cells resumed division. These results suggest that radiation-induced division delay of mammalian cells does not arise from a radiation-induced defect in synthesis and accumulation of microtubule protein.